Inducible expression systems have been developed to facilitate the analysis of gene function in cells and to facilitate the development of effective treatments using gene therapy. These expression systems attempt to control nucleic acid expression by using inducible eukaryotic promoters that are responsive to inducers such as hormones (Lee et al. (1981) Nature 294:228–232; Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038–2042; Klock et al. (1987) Nature 329:734–736; Israel & Kaufman (1989) Nucl. Acids Res. 17:2589–2604); heavy metal ions (Mayo et al. (1982) Cell 29:99–108; Brinster et al. (1982) Nature 296:39–42; Searle et al. (1985) Mol. Cell. Biol. 5:1480–1489); or heat shock (Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L., CRC, Boca Raton, Fla., pp 167–220). However, these expression systems are problematic because the eukaryotic promoters can exhibit a high level of basal expression in the non-induced state; the inducers can promote pleiotropic effects; and the level of induction can be low.
In order to overcome these problems, inducible eukaryotic expression systems utilizing prokaryotic regulatory elements have been developed. The rationale for using prokaryotic regulatory elements in a eukaryotic expression system is based on the theory that effectors modulating the activity of such prokaryotic regulatory elements would not be responsive to eukaryotic cellular components. Therefore, pleiotropic effects would be eliminated. An example of such a system is the lac operator regulatable expression system. In this system, expression of sequences operably linked to the lac operator is constitutively induced (or “turned on”) by a LacR-VP16 fusion protein and is repressed (or “turned off”) in the presence of isopropyl-D-thiogalactopyranoside (IPTG) (Labow et al. (1990), cited supra). In another lac inducible system, the binding of LacR-VP16 to the operator sequence is enhanced by increasing the temperature of the cells. However, IPTG in eukaryotic cells is an inefficient inducer of nucleic acid expression and must be used at concentrations near cytotoxic levels. Furthermore, increasing the temperature of the cells is likely to promote pleiotropic effects in the cells. Thus, there is a need for a more efficient inducible regulatory system that exhibits rapid and high level induction of nucleic acid expression; is highly responsive to a specific exogenous inducer; and exhibits low levels of expression in the uninduced state.
The teteracycline (Tet) inducible system utilizes entirely prokaryotic components and, thus, pleiotropic effects are avoided (Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547–5551; Gossen et al. (1995) Science 268:1766–1769). In this system, the inducer is an integral component of a transactivator that binds to the inducible promoter and drives expression of a nucleic acid of interest. Thus, the intermediate steps in the induction pathway are largely eliminated and the control of expression is highly specific and tightly controlled by contact with the inducer. The Tet-controlled expression system therefore provides an effective means for turning off and turning on nucleic acid expression in cells and, thereby, allowing for the regulated expression of a nucleic acid in cells.
With the advent of functional genomics, large numbers of nucleic acids encoding genes of unknown function have been isolated and cloned. Consequently, there is a critical need to develop rapid and highly efficient methods for screening large numbers of candidate nucleic acids for analysis of gene function and to identify potential targets for development of therapeutic agents.
One approach for studying gene function is to regulate the expression of a nucleic acid in cells and look for a corresponding alteration in cellular phenotype. Consequently, rapid and highly efficient methods of screening diverse populations of cells for an altered phenotype due to the inducible expression of a candidate nucleic acid is highly desirable and useful for the analysis of nucleic acid function and target discovery.
Thus, it is an object of the present invention to provide rapid and highly efficient methods of screening populations of cells for an altered cellular phenotype due to the presence of a candidate bioactive agent, using an inducible expression system that permits the regulated expression of candidate nucleic acids encoding a candidate bioactive agent and permits controlled expression of the candidate nucleic acid at a defined level.